Methods and compositions for enhancing the therapeutic effect of anti-tumor T cells

ABSTRACT

Compositions, e.g., therapeutic agents, and methods are provided for modulating gene and protein expression of Forkhead Box protein 1 (Foxp1). The therapeutic agents include short nucleic acid molecules that modulate gene and protein expression of Forkhead Box protein 1 (Foxp1) expression, viral vectors containing such molecules, T cells transduced with these viruses for adoptive therapies, and any small molecules that bind to and inactivate Foxp1. These compounds and methods have applications in cancer therapy either alone or in combination with other therapies that stimulate the endogenous immune system in the environment of the cancer, e.g., tumor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 14/955,539, filed Dec. 1, 2015, which is adivisional application of U.S. patent application Ser. No. 14/350,588,filed Apr. 9, 2014, now issued as U.S. Pat. No. 9,226,936, which is a371 national stage of International Patent Application No.PCT/US2012/061556, filed Oct. 24, 2012 (expired), which claims thebenefit of the priority of U.S. Provisional Patent Application No.61/552,630, filed Oct. 28, 2011, which applications are incorporatedherein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos.R01CA124515 and 1K22AI070317-01A1 awarded by the National Institutes ofHealth. The government has certain rights in this invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM

Applicant hereby incorporates by reference the Sequence Listing materialfiled in electronic form herewith. This file is labeled“WST128C1US_ST25.txt”.

BACKGROUND OF THE INVENTION

Tumors are imprinted by their immune environment, and this imprintingfacilitates their transformation into populations that can moreeffectively resist the pressure exerted by the subject's immune systemto eradicate them. The immune system can act both positively to inhibitthe progression of tumors and negatively to mold the establishment oftumors that can evade its recognition, or worse to promote theadvancement of tumor development. This process by which the immunesystem can prevent or promote tumor progression is referred to asimmunoediting (Schreiber R D et al, 2011, Science, 331(6024):1565-70;Scarlett U K et al, 2012, J. Exp. Med., 209(3):495-506).

Adoptive cell transfer therapy (ACT) or Adoptive T cell therapy is theex vivo activation, expansion, and subsequent administration oftumor-reactive T cells. Adoptive T cell therapies have focused on theuse of CD8+ T cells, as they have relatively long clonal expansiontimes, can specifically target tumors, and are easily subjected togenetic manipulations. Adoptively transferred tumor-specific T cells canalso be expanded from resected tumors before being geneticallymanipulated. Under ideal circumstances, transferred T cells migrate tothe tumor site and directly lyse tumor cells, while releasing endogenousimmune cells from tumor-induced immunosuppression. However, the tumorenvironment is usually so immunosuppressive that it is difficult toappropriately release these brake mechanisms on antitumor responses.

Adoptive T cell therapy, while highly successful for many nonepithelialcancers, has not yet been effective in the most frequent and aggressiveepithelial cancers, including ovarian carcinoma (see, e.g., Kershaw, M.H. et al. 2006 Clin Cancer Res 12, 6106-15; see also, Dudley M E et al2002 Science 298, 850-4; Bollard, C M et al 2007 Blood, 100:2838-45;Leen, A M et al 2006 Nat. Med., 12:1160-6). Tumor-reactive T cellsproperly conditioned ex vivo have the capacity to induce significanttherapeutic effects against preclinical models of established ovariancancer (Nesbeth Y, S. Y., et al, 2009 Cancer Res. 69, 6331-38; Nesbeth,Y. C. et al. 2010 J Immunol 184, 5654-62). Nevertheless, in thesestudies, anti-tumor T cells did not persist for long periods and,despite a significant survival increase, mice eventually succumbed tothe disease, suggesting that the activity of transplanted T cells wassuboptimal. This lack of success is likely due to the complexities ofthe tumor microenvironment, which apparently causes the rapiddisappearance of transferred lymphocytes. Supporting this proposition,the responsiveness of tumor-reactive T cells in lymph nodes drainingestablished tumors is severely impaired during tumor progression (PMID:22351930).

Further, the ability to produce large numbers of tumor-reactive T cellsis hampered because not only do they usually occur in only lowfrequencies, but also most T cells that robustly respond toself-antigens have either been eliminated during thymic development orrendered nonfunctional by local tolerizing mechanisms.

There remains a need in the art for effective adoptive immunotherapymechanisms for the successful treatment of a variety of cancers.

SUMMARY OF THE INVENTION

In one aspect, a method for enhancing the anti-tumor response in asubject having a cancer, such as a cancer characterized by a solidtumor, involves administering to a subject in need thereof a therapeuticreagent that down-regulates the expression of Foxp1 in T cells.

In another aspect, the method employs a therapeutic reagent thatincludes a short nucleic acid molecule comprising a nucleotide sequencethat is complementary to at least a portion of the nucleotide sequenceencoding FoxP1. In certain embodiments, this short nucleic acid moleculeis a short hairpin RNA (shRNA) or a short interfering RNA (siRNA).

In another aspect, the method employs as the therapeutic agent a plasmidor viral vector that comprises the short nucleic acid molecule, e.g., anshRNA, that comprises a sequence that is complementary to at least aportion of the nucleotide sequence encoding FoxP1, under the control ofregulatory sequences. In another embodiment, the viral vector iscomplexed with a polymer to create a nanoparticle.

In another aspect, the method employs a therapeutic agent that is a Tcell that is transduced or transfected ex vivo with the above-describedviral vector/plasmid. In this method, the T cell is adoptivelytransferred into the subject. In still another embodiment, thetransduced T cell is pulsed with tumor antigen prior to transductionwith the viral vector/plasmid comprising the shRNA sequence that iscomplementary to at least a portion of the nucleotide sequence encodingFoxP1. In still another embodiment, the T cell is transduced ortransfected with a construct that expresses another anti-cancertherapeutic agent, e.g., IL-7.

In another aspect, the method employs a therapeutic agent that is a Tcell pulsed with cancer/tumor-specific antigen, transduced with a vectorexpressing a TCR or chimeric receptor and treated with a zinc-fingernuclease that targets a unique sequence of Foxp1 and removes it from thecells. In this method, the T cell is adoptively transferred into thesubject.

In another aspect, the method employs as the therapeutic agent asynthetic siRNA oligonucleotide comprising a sequence that iscomplementary to at least a portion of the nucleotide sequence encodingFoxP1. In one embodiment, the siRNA is in the form of a nanoparticle.

In still another aspect, the methods described above further involveadministering or co-administering another anti-cancer therapeutic agent,e.g., a chemotherapeutic molecule or a cytokine, e.g., IL-7.

In still another aspect, the methods described above further involveadministering the therapeutic agent before, during or after, surgery toremove or debulk a tumor. In still another aspect, the methods describedabove further involve administering the therapeutic agent before, duringor after, a course of therapeutic radiation. In still another aspect,the methods described above further involve administering thetherapeutic agent before, during or after, a course of chemotherapy.

In still another aspect, a method of preparing a therapeutic compositioncomprises pulsing T cells with a selected cancer antigen ortumor-specific antigen; and transducing said pulsed T cells with avector expressing a construct that down regulates Foxp1, and formulatingsaid pulsed, transfected T cells with a suitable pharmaceutical carrier.

In still another aspect, a therapeutic or prophylactic compositioncomprises a viral vector that targets specifically T cells, the vectorexpressing a construct that inhibits the expression of Foxp1, and apharmaceutically acceptable carrier or diluent. In one embodiment, theconstruct is a short hairpin (shRNA) sequence that suppresses theexpression of Foxp1.

In still another aspect, a therapeutic or prophylactic compositioncomprises a T cell transduced or transfected ex vivo with a viralvector, as described above, wherein the expression of Foxp1 in the Tcell is extinguished or reduced.

Other aspects and advantages of these compositions and methods aredescribed further in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing the results of an experiment described inExample 3 in which anti-tumor Foxp1-deficient T cells (▴) or wild-type Tcells(▾) were adoptively administered to a mouse model of ovarian cancer(ID8-Defb29/Vegf-a). Control mice were treated with PBS (▪). Theadoptive transfer of the anti-tumor Foxp1-deficient T cells to the mousemodel stopped ovarian cancer progression for a prolonged period, so thatall treated mice showed no signs of disease 6 days after the last mousetreated. Survival was 100% at about 73 days, and at 25% at 120 days. Incontrast, administration to the mouse model of PBS or wild-typetumor-reactive T cells resulted in 100% death within 35 or 42 days ofadministration, respectively.

FIG. 1B is a bar graph showing the tumor associated CTLs up-regulateFox-1p during tumor progression, as discussed in Example 2.

FIG. 2 are Western blots quantifying Foxp1 in samples of CD8+T and CD4+T cells and showing that Foxp1 is up-regulated in effector T cells inthe tumor microenvironment: lanes 1 and 4: naïve, negativelyimmunopurified T cell splenocytes, primed against tumor antigen for 7days by incubation with bone marrow-derived dendritic cells pulsed withdouble (UV+gamma) irradiated tumor cells (Day 6 effectors); lanes 2 and5: adoptively transferred (CD45.2+) CD8 cells and CD4 T cells,respectively, FACS-sorted from peritoneal wash samples of CD45.1(congenic) advanced ID8-Defb29/Vegf-a tumor-bearing mice that hadreceived tumor antigen-primed T cells 2 days before sacrifice (Day 2after transfer); and lanes 3 and 6: endogenous (host-derived, CD45.1)tumor-associated T cells FACS-sorted from peritoneal wash samples ofCD45.1 (congenic) advanced ID8-Defb29/Vegf-a tumor-bearing mice that hadreceived tumor antigen-primed T cells 2 days before sacrifice(endogenous, from ascites).

FIG. 3A is a chromatograph trace obtained when naïve, negativelyimmunopurified CD4 T cell splenocytes from wild-type Cre⁻ mice wereCFSE-labeled and expanded in the presence of CD3/CD28 Ab-coated beads(dark line; Invitrogen). When indicated, 5 ng/mL of TGFβ was added(light gray line), and CFSE dilution (indicative of T cellproliferation) was monitored by flow cytometry. Gray shading shows thepresent of a control with no stimulation. Foxp1 T cells are resistant tothe abrogation of T cell expansion induced by TGFβ.

FIG. 3B is a chromatograph trace obtained when naïve, negativelyimmunopurified CD8 splenocytes from wild-type Cre⁻ mice wereCFSE-labeled and expanded in the presence of CD3/CD28 Ab-coated beads(dark line; Invitrogen). When indicated, 5 ng/mL of TGFβ was added(light gray line), and CFSE dilution (indicative of T cellproliferation) was monitored by flow cytometry. Gray shading shows thepresent of a control with no stimulation. Foxp1 T cells are resistant tothe abrogation of T cell expansion induced by TGFβ.

FIG. 3C shows the results of the protocol of FIG. 3A in Fox p1 KO mice.Mice with no Foxp1 T cells display abrogation of T cell expansioninduced by TGFβ.

FIG. 3D shows the results of the protocol of FIG. 3B in Foxp1 KO mice.Mice with no Foxp1 T cells display abrogation of T cell expansioninduced by TGFβ.

FIG. 4 shows Western blot analysis of total and phosphorylated c-Jun inLane 1: naïve, negatively immunopurified, unstimulated CD8 T cellsplenocytes of Foxp1 KO mice; Lane 2: naïve, negatively immunopurified,CD8 T cell splenocytes of Foxp1 KO stimulated for 24 hours in thepresence of CD3/CD28 Ab-coated beads; Lane 3: naïve, negativelyimmunopurified, CD8 T cell splenocytes of Foxp1 KO stimulated in thepresence of CD3/CD28 Ab-coated beads with 5 ng/mL of TGFβ added to thewells; Lane 4: naïve, negatively immunopurified, unstimulated CD8 T cellsplenocytes of wildtype mice; Lane 5: naïve, negatively immunopurified,CD8 T cell splenocytes of wildtype mice stimulated in the presence ofCD3/CD28 Ab-coated beads; Lane 6: naïve, negatively immunopurified, CD8T cell splenocytes of wildtype mice stimulated in the presence ofCD3/CD28 Ab-coated beads with 5 ng/mL of TGFβ added to the wells. TGFβrepresses cJun activation in WT but not in Foxp1 KO CD8+ T cells, asindicated in FIG. 4. Note the induction of c-Jun phosphorylation uponCD3/CD28 activation and how it is selectively abrogated in wild-type Tcells in the presence of TGFβ.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides compositions, e.g., therapeutic agents, andmethods that modulate gene and protein expression of Forkhead Boxprotein 1 (Foxp1) expression. The therapeutic agents include shortnucleic acid molecules that modulate gene and protein expression ofForkhead Box protein 1 (Foxp1) expression, viral vectors containing suchmolecules, T cells transduced with these viruses for adoptive therapies,and any small molecules that bind to and inactivate Foxp1. The compoundsand methods of the present invention have applications in cancer therapyeither alone or in combination with other therapies.

Modulation of the expression of the transcription factor Foxp1 in Tcells promote the engraftment and superior therapeutic activity in thehostile microenvironment of the most aggressive and frequent cancers.The compositions and methods described herein are based on theinventors' finding that down-regulating the expression of FoxP1 inanti-tumor T cells enhances their therapeutic effects in the environmentof a tumor.

The inventors identified that Foxp1 is upregulated in tumormicronevironmental T cells. In addition, Foxp1-deficient anti-tumor Tcells are insensitive to the tolerogenic effect of TGF□, animmunosuppressive mediator universally present in the microenvironmentof virtually all solid tumors. Correspondingly, adoptively transferredFoxp1-deficient tumor-reactive T cells exert significantly superioranti-tumor activity in preclinical models of mice growing establishedorthotropic ovarian tumors.

Definitions

Technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs and by reference to published texts, which provide oneskilled in the art with a general guide to many of the terms used in thepresent application. The following definitions are provided for clarityonly and are not intended to limit the claimed invention.

The term “target nucleic acid” as used herein means any nucleic acidsequence of FoxP1, whose expression or activity is to be modulated. Thetarget nucleic acid can be DNA or RNA.

The term “hairpin” and “stem-loop” can be used interchangeably and referto stem-loop structures. The stem results from two sequences of nucleicacid or modified nucleic acid annealing together to generate a duplex.The loop lies between the two strands comprising the stem. The term“loop” refers to the part of the stem-loop between the two homologousregions (the stem) that can loop around to allow base-pairing of the twohomologous regions. The loop can be composed of nucleic acid (e.g., DNAor RNA) or non-nucleic acid material(s), referred to herein asnucleotide or non-nucleotide loops. A non-nucleotide loop can also besituated at the end of a nucleotide molecule with or without a stemstructure.

The term “complementary” and “complementarity” are interchangeable andrefer to the ability of polynucleotides to form base pairs with oneanother. Base pairs are typically formed by hydrogen bonds betweennucleotide units in antiparallel polynucleotide strands or regions.Complementary polynucleotide strands or regions can base pair in theWatson-Crick manner (e.g., A to T, A to U, C to G). Complete or 100%complementarity refers to the situation in which each nucleotide unit ofone polynucleotide strand or region can hydrogen bond with eachnucleotide unit of a second polynucleotide strand or region.Complementarities less than 100%, e.g., 95%, 90%, 85%, refers to thesituation in which 5%, 10% or 15% of the nucleotide bases of two strandsor two regions of a stated number of nucleotides, can hydrogen bond witheach other.

The term “gene” as used herein means a nucleic acid that encodes a RNAsequence including but not limited to structural genes encoding apolypeptide.

The term “sense region” as used herein means a nucleotide sequence of asmall nucleic acid molecule having complementary to a target nucleicacid sequence. In addition, the sense region of a small nucleic acidmolecule can comprise a nucleic acid sequence having homology with atarget nucleic acid sequence.

The term “antisense region” as used herein means a nucleotide sequenceof a small nucleic acid molecule having a complementarity to a targetnucleic acid sequence. It can also comprise a nucleic acid sequencehaving complementarity to a sense region of the small nucleic acidmolecule.

The term “modulate” or “modulates” means that the expression of the geneor level of RNA molecule or equivalent RNA molecules encoding one ormore protein or protein subunits or peptides, or the expression oractivity of one or more protein subunits or peptides is up regulated ordown regulated such that the expression, level, or activity is greaterthan or less than that observed in the absence of the modulator. Theterm “modulate” includes “inhibit”.

The term “cancer” or “proliferative disease” as used herein means anydisease, condition, trait, genotype or phenotype characterized byunregulated cell growth or replication as is known in the art. It caninclude all types of tumors, lymphomas, carcinomas that can respond tothe modulation of disease-related Fox-1 gene expression in a cell ortissue alone or in combination with other therapies. In variousembodiments of the methods and compositions described herein, the cancercan include, without limitation, breast cancer, lung cancer, prostatecancer, colorectal cancer, brain cancer, esophageal cancer, stomachcancer, bladder cancer, pancreatic cancer, cervical cancer, head andneck cancer, ovarian cancer, melanoma, leukemia, myeloma, lymphoma,glioma, and multidrug resistant cancer.

The term “lymphodepletion” is the elimination of suppressive T cellpopulations, and has been used to enhance the persistence of transferredT cells in vivo. These methods remove cytokine sinks (i.e., endogenouscells that compete with the transferred cells for cytokines that promotetheir activation and function), and through augmenting the function andavailability of APCs, lymphodepletion is thought to enhance theantitumor response.

“Active immunotherapy” is defined as a method in which vaccines such aspeptides, tumor antigens, nucleic acids, engineered tumor cells, ortumor-pulsed DCs are used to activate host antitumor immune cells toreact against a tumor. Active immunotherapy in both mouse and humantumor systems have resulted in potent antitumor responses andregression, and is beneficial in the fact that rather than restrictingresponses to a single epitope/antigen, polyclonal responses can readilybe induced.

“Passive immunotherapy” methods transfer antibodies or antitumorlymphocytes into tumor-bearing hosts to directly induce tumor celldestruction. Passive immunotherapy has revealed high success rates incertain situations; however, as most protocols direct responses againsta single antigen/epitope, and tumors often modulate their expression ofparticular antigens, there is often a high degree of inefficacy.

As used herein, the term “subject”, “patient”, or “mammalian subject”includes primarily humans, but can also be extended to include domesticanimals, such as dogs and cats, and certain valuable animals, such ashorses, farm animals, laboratory animals (e.g., mice, rats) and thelike.

As used herein, the term “antibody,” refers to an immunoglobulinmolecule which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoreactive portionsof intact immunoglobulins. The antibodies useful in the presentinvention may exist in a variety of forms including, for example,polyclonal antibodies, monoclonal antibodies, intracellular antibodies(“intrabodies”), diabodies, Fv, Fab and F(ab)₂, as well as single chainantibodies (scFv), camelid antibodies and humanized antibodies (Harlowet al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, NY; Harlow et al., 1989, Antibodies: A LaboratoryManual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

It should be understood that while various embodiments in thespecification are presented using “comprising” language, under variouscircumstances, a related embodiment is also be described using“consisting of” or “consisting essentially of” language. It is to benoted that the term “a” or “an”, refers to one or more, for example, “ananti-tumor T cell” is understood to represent one or more anti-tumor Tcells. As such, the terms “a” (or “an”), “one or more,” and “at leastone” is used interchangeably herein.

II. COMPOSITIONS USEFUL IN THE INVENTION

As disclosed herein, the compositions described herein modulate theexpression of, or target, Foxp1. The forkhead box (Fox) proteinsconstitute a large transcription factor family with diverse functions indevelopment, cancer and aging. Foxp1, a member of the ‘Foxp’ subfamily,is expressed in many tissues and is a critical transcriptional regulatorin B lymphopoiesis. Conditional deletion of Foxp1 at the CD4+CD8+double-positive (DP) thymocyte stage has proven that Foxp1 is essentialfor the generation of quiescent naive T cells during thymocytedevelopment (Feng et al, 2010 Blood, 115:510-518). In addition, Foxp1expression increases in tumor-associated T cells during cancerprogression.

NCBI Gene ID No. 27086 provides the human gene information for the Foxp1gene of homo sapiens. One transcript variant of the 7102 bp human FOXP1mRNA sequence is reported at NBCI Reference Sequence NM_032682.5 (SEQ IDNO: 1). The protein coding region spans nt 527 through nt 2560 of SEQ IDNO: 1, encoding a 677 amino acid protein SEQ ID NO: 2. Other variantsare known and can be obtained commercially from e.g., GeneCopoeia, amongother commercial sources. Similarly one may obtain murine nucleotide andprotein sequences of FoxP1 from similar sources (see e.g., NCBI Ref Nos.NM_001197322.1, NM_053202.1 and BC064764.1). All such publishedsequences for FoxP1 variants are incorporated herein by reference.

In one embodiment, the compositions and methods described herein targetFoxp1 as set forth in SEQ ID NO: 1. Thus in some embodiments, the term“Foxp1” refers to any Foxp1 protein, peptide, or polypeptide or isoform,including naturally occurring or deliberated mutated or geneticallyengineered sequences, having Foxp1 family activity such as encoded bySEQ ID NO: 1. In other embodiments, the term Foxp1 includes any nucleicacid sequence encoding a Foxp1 protein, peptide, or polypeptide ofmammalian origin, including naturally occurring or deliberated mutatedor genetically engineered sequences. In still other embodiments,Foxp1-related molecules include polymorphisms or single nucleotidepolymorphisms of Foxp1, Foxp1 homologs, and Foxp1 splice and transcriptvariants. Other human isoforms of Foxp1, isoforms 1-8 are identifiedunder the NCBI Gene ID No. 27086.

The compositions described herein can be used to down-regulate Foxp1expression in a subject having a cancer, or more specifically a tumor. Acomposition or molecule that specifically inhibits Foxp1 can block theimmunosuppression of a patient's anti-cancer T cells, and therefore isvery useful in therapy directed at the treatment of tumors.

The compositions useful herein can employ a variety of components and beachieved in multiple ways. Table 1 below sets out the SEQ ID Nos for thesequences discussed hereinbelow. Note that the shRNA sequences in theattached sequence listing are expressed as the DNA sequences used toexpress the shRNA in a vector.

TABLE 1 SEQ ID NO: Sequence Type 3 siRNA for Foxp1 4 siRNA for Foxp1 5shRNA1 for Foxp1 6 Mature sense strand of shRNA1 7 Mature antisensestrand of shRNA1 8 shRNA2 for Foxp1 9 Mature sense strand, shRNA2 10Mature antisense strand, shRNA2 11 shRNA3 for Foxp1 12 Mature sensestrand, shRNA3 13 Mature antisense strand, shRNA3 14 shRNA4 for Foxp1 15Mature sense strand, shRNA4 16 Mature antisense strand, shRNA4 17 shRNA5for Foxp1 18 Mature sense strand, shRNA5 19 Mature antisense strand,shRNA5 20 shRNA6 for Foxp1 21 Mature sense strand, shRNA6 22 Matureantisense strand, shRNA6 23 shRNA7 for Foxp1 24 Mature sense strand,shRNA7 25 Mature antisense strand, shRNA7 26 shRNA8 for Foxp1 27 Maturesense strand, shRNA8 28 Mature antisense strand, shRNA8 29 shRNA9 forFoxp1 30 Mature sense strand, shRNA9 31 Mature antisense strand, shRNA932 shRNA10 for Foxp1 33 Mature sense strand, shRNA10 34 Mature antisensestrand, shRNA10 35 shRNA11 for Foxp1 36 Mature sense strand, shRNA11 37Mature antisense strand, shRNA11 38 shRNA12 for Foxp1 39 Mature sensestrand, shRNA12 40 Mature antisense strand, shRNA12 41 shRNA13 for Foxp142 Mature sense strand, shRNA13 43 Mature antisense strand, shRNA13 44shRNA14 for Foxp1 45 Mature sense strand, shRNA14 46 Mature antisensestrand, shRNA14 47 shRNA15 for Foxp1 48 Mature sense strand, shRNA15 49Mature antisense strand, shRNA15 50 shRNA16 for Foxp1 51 Mature sensestrand, shRNA16 52 Mature antisense strand, shRNA16 53 shRNA17 for Foxp154 Mature sense strand, shRNA17 55 Mature antisense strand, shRNA17

Short Nucleic Acid Molecules

A short nucleic acid molecule useful in the compositions and in themethods described herein is any nucleic acid molecule capable ofinhibiting or down-regulating FoxP1 gene expression. Typically, shortinterfering nucleic acid molecules are composed primarily of RNA, andinclude siRNA or shRNA, as defined below. A short nucleic acid moleculemay, however, include nucleotides other than RNA, such as in DNAi(interfering DNA), or other modified bases. Thus, the term “RNA” as usedherein means a molecule comprising at least one ribonucleotide residueand includes double stranded RNA, single stranded RNA, isolated RNA,partially purified, pure or synthetic RNA, recombinantly produced RNA,as well as altered RNA such as analogs or analogs of naturally occurringRNA. In one embodiment the short nucleic acid molecules of the presentinvention is also a short interfering nucleic acid (siNA), a shortinterfering RNA (siRNA), a double stranded RNA (dsRNA), a micro RNA(.mu.RNA), and/or a short hairpin RNA (shRNA) molecule. The shortnucleic acid molecules can be unmodified or modified chemically.Nucleotides of the present invention can be chemically synthesized,expressed from a vector, or enzymatically synthesized.

In some embodiments, the short nucleic acid comprises between 18 to 60nucleotides. In another embodiment, the short nucleic acid molecule is asequence of nucleotides between 25 and 50 nucleotides in length. Instill other embodiments, the short nucleic acid molecule ranges up to 35nucleotides, up to 45, up to 55 nucleotides in length, depending uponits structure. These sequences are designed for better stability andefficacy in knockdown (i.e., reduction) of Foxp1 gene expression. In oneembodiment, the nucleic acid molecules described herein comprises 19-30nucleotides complementary to a Foxp1 nucleic acid sense sequence,particularly an open reading frame of Foxp1. In one embodiment, thenucleic acid molecules described herein comprises 19-30 nucleotidescomplementary to a Foxp1 antisense nucleic acid sequence strand. In oneembodiment, the nucleic acid molecules described herein comprises 19-30nucleotides complementary to a Foxp1 nucleic acid sense sequence andcomprises 19-30 nucleotides complementary to a Foxp1 antisense nucleicacid sequence strand.

1. siRNA Molecules

In one embodiment, a useful therapeutic agent is a small interfering RNA(siRNA) or a siRNA nanoparticle. siRNAs are double stranded, typically21-23 nucleotide small synthetic RNA that mediate sequence-specific genesilencing, i.e., RNA interference (RNAi) without evoking a damaginginterferon response. siRNA molecules typically have a duplex region thatis between 18 and 30 base pairs in length. FoxP1 siRNAs are designed tobe homologous to the coding regions of FoxP1 mRNA (e.g., SEQ ID NO: 1)and suppress gene expression by mRNA degradation. The siRNA associateswith a multi protein complex called the RNA-induced silencing complex(RISC), during which the “passenger” sense strand is enzymaticallycleaved. The antisense “guide” strand contained in the activated RISCthen guides the RISC to the corresponding mRNA because of sequencehomology and the same nuclease cuts the target mRNA, resulting inspecific gene silencing. The design of si/shRNA preferably avoids seedmatches in the 3′UTR of cellular genes to ensure proper strand selectionby RISC by engineering the termini with distinct thermodynamicstability. A single siRNA molecule gets reused for the cleavage of manytarget mRNA molecules. RNAi can be induced by the introduction ofsynthetic siRNA.

In one embodiment, a siRNA molecule of the invention comprises a doublestranded RNA wherein one strand of the RNA is complimentary to the RNAof Foxp1. In another embodiment, a siRNA molecule of the inventioncomprises a double stranded RNA wherein one strand of the RNA comprisesa portion of a sequence of RNA having Foxp1 sequence. SEQ ID Nos: 3 and4 illustrate two exemplary siRNAs for Foxp1. Synthetic siRNA effects areshort lived (a few days) probably because of siRNA dilution with celldivision and also degradation.

In one embodiment, siRNA without any chemical modification having highstability and specificity for Foxp1, are useful as therapeutics alone,or in combination with other therapies for cancer. In anotherembodiment, siRNA oligonucleotides targeting Foxp1 are complexed orconjugated to a polymer or any other material that stabilizes siRNA, foruse as therapeutics alone, or in combination with other therapies forcancer.

Among such stabilizing polymers and materials are polyethyleneimine(PEI), which may be conjugated to siRNA, resulting in the generation ofnanocomplexes of about 50 nm, as described in Cubillos-Ruiz J R, et al,2009 J. Clin. Invest., 119(8):2231-44, incorporated by reference herein.In another embodiment, such a stabilizing material is chitosan. In oneembodiment, the siRNA is in a stable composition, with or withoutconjugation, with cholesterol. In still other embodiments, siRNA may becombined with conjugates such as a lipid, a cationic lipid, aphospholipid, and a liposome.

In another embodiment, the siRNA is in a stable composition, with orwithout conjugation, to an antibody or fragment thereof that permits thesiRNA to be preferentially targeted. In one embodiment, the antibody isan antibody or fragment to a desirable molecule, such as an IL7receptor. In another embodiment, the antibody is an antibody or fragmentto a T cell surface marker, a T cell receptor or a chimeric receptorwhich also permits targeting. For example, in one another embodiment,the siRNA are linked to thiolated F(ab)2 fragments of monoclonalantibodies targeting T cell surface markers (e.g., CD3, CTLA4, CD44,CD69 or CD25).

In another embodiment, the antibody or fragment is to a T cell receptoror chimeric receptor. T cell receptors or chimeric receptors forassociation with, or co-expression with the siRNA include withoutlimitation, TCRs against human antigens. Among such useful TCRs includethose that have been transduced in adoptively transferred T cells(reviewed in Trends Biotechnol. 2011 November; 29(11):550-7). In oneembodiment, the TCR is the receptor that binds human carcinoembryonicantigen (Parkhurst M R et al, 2011 Mol. Ther., 19(3):620-6), NY-ESO-1(Robbins P F et al, 2011 J. Clin. Oncol., 29(7):917-24), MAGE-A3(Chinnasamy N et al 2011 J. Immunol., 186(2):685-96) and MART-1, gp100and p53 (Morgan R A et al, 2006 Science, 314(5796):126-9). Associationwith such TCRs is described in Westwood et al, 2005, cited herein.

Examples of chimeric receptors useful in the compositions and methodsdescribed herein are chimeric receptors against the antigens CD19 (KolosM, et al, 2011 Sci Transl. Med., 3(95):95ra73) and Epstein Barr virus(Fondell, J D et al, 1990 J. Immunol., 144(3):1094-103). Other chimericreceptors have also targeted mesothelin (Moon E K et al, 2011 ClinCancer Res., 17(14):4719-30) and the folate receptor (Song D G et al,2011 Cancer Res., 71(13):4617-27).

2. shRNA Molecules

In another embodiment, the short nucleic acid molecule is a smallhairpin RNA (shRNA). An shRNA molecule useful in the methods andcompositions described herein is generally defined as an oligonucleotidecontaining the about 18-23 nucleotide siRNA sequence followed by a ˜9-15nt loop and a reverse complement of the siRNA sequence. The loopnucleotides generally form a non-coding sequence. Examples ofcommercially available shRNA sequences targeting human Foxp1 include thefollowing DNA sequences along with their mature and antisense strandslisted in Table 1. These DNA sequences, when expressed in a vector, formthe corresponding shRNA sequences. For example, as demonstrated inshRNA1 for Foxp1 SEQ ID NO: 5, nucleotide positions 21-39 are the maturesense strand, followed by the sequence of nucleotides 42-57 containingthe loop and then followed by nucleotides 61-79, which are the antisensestrand. The same analysis can be made of the other shRNA sequences inTable 1. Thus, other embodiments of shRNAs include those of SEQ ID NOs:5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50 and 53, ortheir mature or antisense strands.

shRNAs can be cloned in plasmids or in non-replicating recombinant viralvectors to endogenously/intracellularly express shRNA, which issubsequently processed in the cytoplasm to siRNA. The shRNA effects arelonger lasting because they are continually produced within the cellsand thus have an effect that lasts the duration of the cell's life.

B. Recombinant Vectors Carrying an shRNA Construct

As used herein, a vector may include any genetic element including,without limitation, naked DNA, a phage, transposon, cosmid, episome,plasmid, bacteria, or a virus. As used herein, the term vector refers toa genetic element which expresses, or causes to be expressed, thedesired construct that inhibits the expression of Foxp1 in the targetcell ex vivo or in vivo.

These shRNAs can be produced in plasmid based systems, of which many arecommercially available. However, because they are easy to deliver,non-replicating recombinant viral vectors are commonly used for shRNAexpression. Thus, in one embodiment, the vector is a non-pathogenicvirus. In another embodiment, the vector is a non-replicating virus. Inone embodiment, a desirable viral vector may be a retroviral vector,such as a lentiviral vector. In another embodiment, a desirable vectoris an adenoviral vector. In still another embodiment, a suitable vectoris an adeno-associated viral vector. Adeno, adeno-associated andlentiviruses are generally preferred because they infect activelydividing as well as resting and differentiated cells such as the stemcells, macrophages and neurons. A variety of adenovirus, lentivirus andAAV strains are available from the American Type Culture Collection,Manassas, Va., or available by request from a variety of commercial andinstitutional sources. Further, the sequences of many such strains areavailable from a variety of databases including, e.g., PubMed andGenBank.

In one embodiment, a lentiviral vector is used. Among useful vectors arethe equine infectious anemia virus and feline as well as bovineimmunodeficiency virus, and HIV-based vectors. A variety of usefullentivirus vectors, as well as the methods and manipulations forgenerating such vectors for use in transducing cells and expressingheterologous genes, e.g., the shRNA that inhibits the expression ofFoxp1, are described in N Manjunath et al, 2009 Adv Drug Deliv Rev.,61(9): 732-745, incorporated herein by reference. In one embodiment theself-inactivating lentiviral vector (GeMCRIS 0607-793) which wassuccessfully used to transduce T cells directed against tumor cells inleukemia patients (Porter et al., N Engl J Med. 2011 Aug. 25;365(8):725-33) is useful to carry and express a nucleotide sequence,e.g., an shRNA, that inhibits the expression of Foxp1. See thedescription of one such desirable vector in Example 5 below.

In another embodiment, the vector used herein is an adenovirus vector.Such vectors can be constructed using adenovirus DNA of one or more ofany of the known adenovirus serotypes. See, e.g., T. Shenk et al.,Adenoviridae: The Viruses and their Replication”, Ch. 67, in FIELD'SVIROLOGY, 6th Ed., edited by B. N Fields et al, (Lippincott RavenPublishers, Philadelphia, 1996), p. 111-2112; U.S. Pat. No. 6,083,716,which describes the genome of two chimpanzee adenoviruses; U.S. Pat. No.7,247,472; WO 2005/1071093, etc. One of skill in the art can readilyconstruct a suitable adenovirus vector to carry and express a nucleotidesequence as described herein, e.g., a shRNA that inhibits the expressionof Foxp1, by resort to well-known publications and patents directed tosuch viral vectors. See, e.g., Arts, et al, 2003 Adenoviral vectors forexpressing siRNAs for discovery and validation of gene function, GenomeResearch, 13:2325-32.

In another embodiment, the vector used herein is an adeno-associatedvirus vector. In another embodiment, the vector used herein is anadeno-associated virus (AAV) vector. Such vectors can be constructedusing AAV DNA of one or more of the known AAV serotypes. See, e.g., U.S.Pat. No. 7,906,111 (Wilson); Gao et al, Novel Adeno-Associated VirusesFrom Rhesus Monkeys as Vectors for Human Gene Therapy, PNAS, vol. 99,No. 18, pp. 11854-11859, (Sep. 3, 2002); Rutledge et al, InfectiousClones and Vectors Derived from Adeno-Associated Virus (AAV) SerotypesOther Than AAV Type 2, Journal of Virology, vol. 72, pp. 309-319,(January 1998). One of skill in the art can readily construct a suitableAAV vector to carry and express a nucleotide sequence as describedherein, e.g., an shRNA that inhibits the expression of Foxp1, by resortto well-known publications and patents directed to such AAV vectors.See, e.g, Grimm et al, Adeno-associated virus vectors for short hairpinRNA expression, Methods Enzymology, 392, 381-405 (2005); U.S. Pat. Nos.7,803,611; 7,696,179.

In yet another embodiment, the vector used herein is a bacterial vector.In one embodiment, the bacterial vector is Listeria monocytogenes.Listeria monocytogenes is a food borne pathogen which has been found tobe useful as a vaccine vehicle, especially in attenuated form. See,e.g., Ikonomidis et al, J. Exp. Med, 180:2209-18 (December 1994); Laueret al, Infect. Immunity, 76(8):3742-53 (August 2008). Listeriamonocytogenes are known to spontaneously infect dendritic cells,listerial adhesion factors internalin A and internalin B (Kolb-Mäurer etal, Infection & Immunity, 68(6):3680-8 (June 2000)). Thus, in oneembodiment, the bacterial vector is live-attenuated or photochemicallyinactivated. The heterologous gene of interest, e.g., the shRNA the caninhibit Foxp1, such as those listed in Table 1, can be expressedrecombinantly by the bacteria, e.g., via a plasmid introduced into thebacteria, or integrated into the bacterial genome, i.e., via homologousrecombination.

Generally, each of these vectors also comprises a minigene. By“minigene” is meant the combination of a selected nucleotide sequence(e.g., a short nucleic acid sequence described herein or shRNA ofTable 1) and the operably linked regulatory elements necessary to drivetranslation, transcription and/or expression of the gene product in thehost cell in vivo or in vitro. As used herein, “operably linked”sequences include both expression control sequences that are contiguouswith the gene of interest and expression control sequences that act intrans or at a distance to control the gene of interest.

These vectors also include conventional control elements that permitstranscription, translation and/or expression of the shRNA in a celltransfected with the plasmid vector or infected with the viral vector. Agreat number of expression control sequences, including promoters whichare native, constitutive, inducible and/or tissue-specific, are known inthe art and may be utilized. In one embodiment, the promoter is an RNApolymerase promoter. In another embodiment, the promoter is an RNApolymerase promoter selected from U6, H1, T7, pol I, pol II and pol IIIpromoters. In another embodiment, the promoter is a constitutivepromoter. In another embodiment, the promoter is an inducible promoter.In one embodiment, the promoter is selected based on the chosen vector.In another embodiment, when the vector is lentivirus, the promoter isU6, H1, CMV IE gene, EF-1α, ubiquitin C, or phosphoglycero-kinase (PGK)promoter. In another embodiment, when the vector is an AAV, the promoteris an RSV, U6, or CMV promoter. In another embodiment, when the vectoris an adenovirus, the promoter is RSV, U6, CMV, or H1 promoters. Inanother embodiment, when the vector is Listeria monocytogenes, thepromoter is a hly or actA promoter.

Still other conventional expression control sequences include selectablemarkers or reporter genes, which may include sequences encodinggeneticin, hygromicin, ampicillin or purimycin resistance, among others.Other components of the vector may include an origin of replication.Selection of these and other promoters and vector elements areconventional and many such sequences are available [see, e.g., Sambrooket al, and references cited therein].

These vectors are generated using the techniques and sequences providedherein, in conjunction with techniques known to those of skill in theart. Such techniques include conventional cloning techniques of cDNAsuch as those described in texts (Sambrook et al, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.),use of overlapping oligonucleotide sequences, polymerase chain reaction,and any suitable method which provides the desired nucleotide sequence.

Thus, in one embodiment, using the information taught herein andpublically available and known vector construction components andtechniques, one of skill in the art can construct a viral vector (orplasmid) that expresses the desired construct, e.g., a short hairpin(shRNA) sequence that suppresses the expression of Foxp1. In certainembodiments, the DNA sequence which upon expression in a vectorexpresses the shRNA, is selected from those reported in Table 1. See,the vector of Example 5.

In still another embodiment, the vector may be designed to co-expressmore than one shRNA that suppresses the expression of Foxp1, e.g., suchas more than one of the sequences of Table 1.

In yet another embodiment, the vector may be designed to co-express aconstruct that enables targeting of the virus vector to only T cells.Such targeting will enable the virus to be employed in vivo. Forexample, the virus vector is designed to co-express an anti-tumor T cellreceptor or a chimeric anti-tumor T cell receptor, or portion of anantibody or fragment to a T cell surface marker. Among suitableconstructs for co-expression are fragments of monoclonal antibodiestargeting T cell surface markers (e.g., CD3, CTLA4, CD44, CD69 or CD25),TCRs against human antigens, such as human carcinoembryonic antigen,NY-ESO-1, MAGE-A3 and MART-1, gp100 and p53. Chimeric receptors that maybe co-expressed include, e.g., chimeric receptors against the antigensCD19, Epstein Barr virus, mesothelin and the folate receptor.

For example, by using the above-noted lentiviral vector (GeMCRIS0607-793) and transductions at a multiplicity of infection of 5, a highlevel of expression of chimeric receptors directed against tumor cellantigens can be obtained in >85% primary human T cells (Milone et al.,Molecular Therapy (2009) 17 8, 1453-1464). In one embodiment, a minigeneor cassette containing a Foxp1 shRNA sequence downstream of a RNApolymerase III promoter (e.g., the H1 or the U6 promoters) could be subcloned into the same lentiviral vector, which would therefore conferexpression of the chimeric receptor and silencing of Foxp1 in the same Tcell.

In still other embodiments, the viral vectors or plasmids carrying theFoxp1 shRNA are complexed or conjugated to a polymer or any othermaterial that stabilizes the vector or assists in its targeting. Amongsuch stabilizing polymers and materials are polyethyleneimine (PEI),which may be conjugated to the vector, resulting in the generation ofnanocomplexes of about 50 nm, as described in Cubillos-Ruiz J R, et al,2009 J. Clin. Invest., 119(8):2231-44, incorporated by reference herein.In another embodiment, such a stabilizing material is chitosan. In oneembodiment, the vector is in a stable composition, with or withoutconjugation, with cholesterol. In another embodiment, the vector may beconjugated, to an antibody or fragment thereof that permits the vectorto be preferentially targeted.

In one embodiment, the antibody is an antibody or fragment to adesirable molecule, such as a IL7 receptor. In another embodiment, theantibody is an antibody or fragment to a T cell surface marker, a T cellreceptor or a chimeric receptor which also permits targeting. Forexample, in one another embodiment, the vectors are linked to thiolatedF(ab)2 fragments of monoclonal antibodies targeting T cell surfacemarkers (e.g., CD3, CTLA4, CD44, CD69 or CD25). In another embodiment,the antibody or fragment is to a T cell receptor or chimeric receptorsuch as those described above.

C. Anti-Tumor T Cells for Adoptive Transfer

To generate cells for adoptive transfer, the above-described vectorscarrying the minigene expressing at least one Foxp1 shRNA, andoptionally a second construct for co-expression, are delivered to ananti-tumor T cell. “Anti-tumor T cells” are primarily, but notexclusively, CD8 (cytotoxic) T cells with activity against an autologoustumor, which are able to become activated and expand in response toantigen. Anti-tumor T cells, useful for adoptive T cell transferinclude, in one embodiment, peripheral blood derived T cells geneticallymodified with receptors that recognize and respond to tumor antigens.Such receptors are generally composed of extracellular domainscomprising a single-chain antibody (scFv) specific for tumor antigen,linked to intracellular T cell signaling motifs (see, e.g., Westwood, J.A. et al, 2005, Proc. Natl. Acad. Sci., USA, 102(52):19051-19056). Otheranti-tumor T cells include T cells obtained from resected tumors. Inanother embodiment, the T cell is a polyclonal or monoclonaltumor-reactive T cell, i.e., obtained by apheraesis, expanded ex vivoagainst tumor antigens presented by autologous or artificialantigen-presenting cells. In another embodiment, the T cell isengineered to express a T cell receptor of human or murine origin thatrecognizes a tumor antigen.

In one embodiment, T cells are designed for autologous adoptive transferinto cancer patients. The T cells are engineered ex vivo to express ashRNA capable of down-regulating FoxP1 expression once the T cells aredelivered to the subject. In another embodiment, the subject's T cellscan be manipulated in vivo by administration of certain therapeuticagents designed to downregulate Foxp1 activity. In one embodiment, sucha therapeutic agent is a virus engineered to express the shRNA.Generally, when delivering the vector comprising the minigene bytransfection to the T cells, the vector is delivered in an amount fromabout 5 μg to about 100 μg DNA to about 1×10⁴ cells to about 1×10¹³cells. In another embodiment, the vector is delivered in an amount fromabout 10 to about 50 μg DNA to 1×10⁴ cells to about 1×10¹³ cells. Inanother embodiment, the vector is delivered in an amount from about 5 μgto about 100 μg DNA to about 10⁵ cells. However, the relative amounts ofvector DNA to the T cells may be adjusted, taking into considerationsuch factors as the selected vector, the delivery method and the hostcells selected. The vector may be introduced into the T cells by anymeans known in the art or as disclosed above, including transfection,transformation and infection. The heterologous gene of interest, e.g.,the Foxp1 shRNA, may be stably integrated into the genome of the hostcell, stably expressed as episomes, or expressed transiently.

In still another embodiment, the T cells are primed/pulsed with andagainst a selected cancer, or tumor-specific, antigen, or with andagainst multiple tumor antigens before transfection with the vectorcarrying the Foxp1 shRNA. In another example, polyclonal T cells primedagainst multiple tumor antigens are transduced with the above-describedlentiviral vector encoding a Foxp1 shRNA sequence. These adoptive Tcells are prepared by pulsing T cells with a selected cancer, ortumor-specific, antigen; transducing the pulsed T cells with a vectorexpressing a construct that down regulates Foxp1, and formulating saidpulsed, transfected T cells with a suitable pharmaceutical carrier.

In another embodiment, the anti-tumor T cells are prepared for adoptivetherapy by pulsing/priming the T cells with or against a selectedcancer, or tumor-specific, antigen. The pulsed cells are then transducedwith a vector expressing a TCR or chimeric anti-tumor receptor. Thesecells are then treated ex vivo with zinc-finger nucleases with sequencesthat are optimized and designed to target the unique sequence of Foxp1.By taking advantage of endogenous DNA repair machinery, these reagentsremove Foxp1 from the anti-tumor T cells (lymphocytes) before adoptivetransfer. The resulting T cells are prepared for adoptive therapy in asuitable pharmaceutical carrier. These T cells are prepared usingtechniques described in the comparable deletion of CCR5 in T cellsadministered to HIV infected patients in Perez et al, Nat. Biotechnol.2008; 26:808-16, incorporated by reference herein.

Alternatively, the T cells can be transfected with multiple differentviral vectors that express different Foxp1 shRNAs, and/or express TCRsand Foxp1 shRNA and/or chimeric receptors and Foxp1RNA, using the sametechniques as described above.

D. Small Molecules

In still another embodiment, such a therapeutic agent is a smallmolecule or drug that binds to FoxP1 and inhibits its function.

The compositions comprising the small nucleic acid molecules, viruses,plasmids or T cells described above may be further associated with apharmaceutically acceptable carrier for in vivo delivery. As used hereinthe term “pharmaceutically acceptable carrier” or “diluent” is intendedto include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with administration to humans. In oneembodiment, the diluent is saline or buffered saline.

II. METHODS

All of the compositions and components described above may be used inthe methods described herein for stimulating anti-tumor immune activity.Thus, in one embodiment, a method of treating a cancer or enhancing ananti-tumor response in a subject having a cancer involves administeringto a subject in need thereof a therapeutic reagent that down-regulatesthe expression of Foxp1 in T cells within the environment of the cancercells. In one embodiment, the cancer is characterized by the presence ofa solid tumor and the expression of Foxp1 is desirably down-regulated inT cells within the tumor microenvironment. These methods areparticularly use for enhancing the treatment of cancer, particularlycancers that are not sensitive to other conventional treatments. Incertain embodiments, the cancer is breast cancer, lung cancer, prostatecancer, colorectal cancer, brain cancer, esophageal cancer, stomachcancer, bladder cancer, pancreatic cancer, cervical cancer, head andneck cancer, ovarian cancer, melanoma, leukemia, myeloma, lymphoma,glioma, or multidrug resistant cancer.

In one embodiment, the method involves administering a short nucleicacid molecule which may be a short hairpin RNA (shRNA), a shortinterfering RNA (siRNA), a double stranded RNA (dsRNA), a micro RNA, andan interfering DNA (DNAi) molecule to the subject or carried within theanti-tumor T cell. In one embodiment, the siRNA is delivered with adelivery agent, such as a lipid, a cationic lipid, a phospholipid, and aliposome to carry siRNA oligonucleotides targeting the expression ofFoxp1. In certain embodiments, the synthetic siRNA oligonucleotide is inthe form of a nanoparticle complexed with a polymer or other material asdescribed in detail above. In one embodiment, the reagent is any of theshort nucleic acid molecules of the present invention. In anotherembodiment, the short nucleic acid molecule is between 19 to 65nucleotides. In yet another embodiment, the short nucleic acid moleculecomprises 19-30 nucleotides that are complementary to a sequence withina full length Foxp1 nucleotide sequence SEQ ID NO: 1. In one embodiment,the short nucleic acid molecule comprises an siRNA with the sequence ofSEQ ID NO: 3 or 4. In another embodiment, the short nucleic acidmolecule comprises a shRNA with a sequence selected from the groupconsisting of SEQ ID NOs: 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38,41, 44, 47, 50 and 53.

In another embodiment, the method provides administering a vector suchas those described in detail above, which specifically infected only Tcells, and which express a construct that inhibits the expression ofFoxp1, in a pharmaceutically acceptable carrier or diluent. In oneembodiment, where the method the use of a viral vector comprising ashort nucleic acid molecule, anti-tumor T cells in the tumor environmentare infected by said virus in vivo and Foxp1 is down regulated in theinfected T cells. For this embodiment, the virus specifically infectsonly T cells. In another embodiment, a plasmid or viral vector comprisesthe short nucleic acid molecule, under the control of regulatorysequences. In one embodiment, the viral vector is selected from thegroup consisting of adenovirus or lentivirus. In another embodiment, theviral vector is complexed with a polymer. In one embodiment, the polymeris PEI, chitosan or any other material that stabilizes siRNA. In anotherembodiment, the method provides administering a viral vector thatco-expresses an anti-tumor T cell receptor or a chimeric anti-tumor Tcell receptor. Anti-tumor T cells in the tumor environment becomeinfected by said virus in vivo and Foxp1 is down regulated in theinfected T cells.

In another embodiment, the method involves adoptive T cell therapy andinvolves administering an anti-tumor T cell as described in detailabove, e.g., a T cell transduced or transfected ex vivo with the viralvector, wherein the expression of Foxp1 in the T cell is extinguished orreduced. As described above, in one embodiment, the viral vector/plasmidis transduced ex vivo into a T cell and said T cell is introduced intothe subject. In one embodiment, the construct is administered ex vivo toa T cell selected from the group consisting of (a) apolyclonal/monoclonal tumor-reactive T cell, (b) a tumor-infiltratinglymphocyte generated from aphaeresis samples or isolated from a tumor ofa cancer patient, and (c) a T cell conditioned for adoptive transfer. Inone embodiment, the T cell is pulsed with tumor antigen prior totransduction with the viral vector/plasmid. In another embodiment, the Tcell has been conditioned for adoptive transfer by pulsing ex vivo witha tumor-specific antigen before it is transduced with the virus vector.In still another embodiment, the virus stably expresses the construct inthe T cell. Expression of the construct in the T cells transduced exvivo increases anti-tumor immunity upon administration to the subject.

Down-regulating Foxp1 in anti-tumor T cells (conditioned for adoptivetransfer or not) enhances the therapeutic activity of the T cells andprolong the survival of cancer patients. The therapeutic compositionsadministered by these methods, e.g., whether virus, virus nanoparticle,siRNA alone, siRNA nanoparticle, anti-tumor T cell treated for adoptivetherapy, are administered directly into the environment of the cancercell or tumor microenvironment of the subject. Conventional andpharmaceutically acceptable routes of administration include, but arenot limited to, systemic routes, such as intraperitoneal, intravenous,intranasal, intravenous, intramuscular, intratracheal, subcutaneous, andother parenteral routes of administration or intratumoral or intranodaladministration. Routes of administration may be combined, if desired. Insome embodiments, the administration is repeated periodically.

These therapeutic compositions may be administered to a patient,preferably suspended in a biologically compatible solution orpharmaceutically acceptable delivery vehicle. The various components ofthe compositions are prepared for administration by being suspended ordissolved in a pharmaceutically or physiologically acceptable carriersuch as isotonic saline; isotonic salts solution or other formulationsthat will be apparent to those skilled in such administration. Theappropriate carrier will be evident to those skilled in the art and willdepend in large part upon the route of administration. Other aqueous andnon-aqueous isotonic sterile injection solutions and aqueous andnon-aqueous sterile suspensions known to be pharmaceutically acceptablecarriers and well known to those of skill in the art may be employed forthis purpose.

The viral vectors or siRNA nanoparticles are administered in sufficientamounts to transduce the targeted T cells and to provide sufficientlevels of gene transfer and expression to reduce or inhibit expressionof Foxp1 and provide a therapeutic benefit without undue adverse or withmedically acceptable physiological effects, which can be determined bythose skilled in the medical arts. The adoptive T cells are similarlyadministered to express the Foxp1 shRNA and to reduce or inhibitexpression of Foxp1 to provide a therapeutic benefit without undueadverse or with medically acceptable physiological effects, which can bedetermined by those skilled in the medical arts.

Dosages of these therapeutic reagents will depend primarily on factorssuch as the condition being treated, the age, weight and health of thepatient, and may thus vary among patients. For example, atherapeutically effective adult human or veterinary dosage of the viralvector or siRNA nanoparticle is generally in the range of from about 100μL to about 100 mL of a carrier containing concentrations of from about1×10⁶ to about 1×10¹⁵ particles, about 1×10¹¹ to 1×10¹³ particles, orabout 1×10⁹ to 1×10¹² particles virus. Methods for determining thetiming of frequency (boosters) of administration will include anassessment of tumor response to the vector administration. As anotherexample, the number of adoptively transferred anti-tumor T cells can beoptimized by one of skill in the art depending upon the response andoverall physical health and characteristics of the individual patient.

In one embodiment, such a dosage can range from about 10⁵ to about 10¹¹cells per kilogram of body weight of the subject. In another embodiment,the dosage of anti-tumor T cells is about 1.5×10⁵ cells per kilogram ofbody weight. In another embodiment, the dosage of anti-tumor T cells isabout 1.5×10⁶ cells per kilogram of body weight. In another embodiment,the dosage of anti-tumor T cells is about 1.5×10⁷ cells per kilogram ofbody weight. In another embodiment, the dosage of anti-tumor T cells isabout 1.5×10⁸ cells per kilogram of body weight. In another embodiment,the dosage of anti-tumor T cells is about 1.5×10⁹ cells per kilogram ofbody weight. In another embodiment, the dosage of anti-tumor T cells isabout 1.5×10¹⁰ cells per kilogram of body weight. In another embodiment,the dosage of anti-tumor T cells is about 1.5×10¹¹ cells per kilogram ofbody weight. Other dosages within these specified amounts are alsoencompassed by these methods. See, e.g., Dudley et al, 2002, citedabove; and Porter et al, 2011, cited above.

In still other embodiments, these methods of down-regulating FoxP1 arepart of a combination therapy. In one embodiment, the short nucleic acidmolecules, such as siRNA and shRNA, the viral vectors, and theanti-tumor T cells prepared for adoptive immunotherapy as describedabove, can be administered alone or in combination with various othertreatments or therapies for the cancer.

In one embodiment, the methods include IL-7 treatment as tumor-specifichost conditioning strategies together with Foxp1-deficient T celltransfer. IL-7Rα is one of the most critical cytokine receptors for Tcell survival. The IL-7R complex is composed of IL-7Rα and the commoncytokine receptor γ-chain (γ_(c)), but control of IL-7 signaling isprimarily dependent on the regulation of IL-7Rα (Mazzucchelli & Durum,2007, Nat. Rev. Immunol., 7:144-54; Jiang Q et al 2005 Cytokine GrowthFactor Rev., 16:513-33). As we demonstrated, Foxp1 represses IL-7Rαexpression, and Foxp1-deficient naive CD8⁺ T cells even proliferate inresponse to IL-7 directly in the absence of overt TCR stimulation.Through the administration of IL-7, we will take advantage of the higherIL-7R expression in Foxp1-deficient T cells to promote their in vivoactivity, expansion and survival after adoptive transfer. Therefore,administration of IL-7 is a synergistic host conditioning strategytogether with the adoptive transfer of anti-tumor Foxp1-deficient Tcells. Foxp1-deficient naive T cells will show superior proliferation toIL-7, compared to wild-type activated T cells. Exogenous administrationof IL-7 further promotes the in vivo activity specifically ofFoxp1-deficient T cells.

Thus, in one embodiment, the method further comprises co-administeringexogenous IL-7 to the subject to promote the in vivo activity ofFoxp1-deficient T cells. In another embodiment, the therapeutic agentthat down-regulates Foxp1, whether the siRNA, nanoparticle, virus ortransduced T cells, is provided in combination with a short nucleic acidmolecule that targets IL7 Receptor. This molecule can be co-expressed inthe vector or in the anti-tumor T cell for adoptive therapy.

In another embodiment, the method further comprises administering to thesubject along with the therapeutic agent the down-regulates Foxp1, anadjunctive anti-cancer therapy which may include a monoclonal antibody,chemotherapy, radiation therapy, a cytokine, or a combination thereof.These therapies may include co-expression of T cell receptor proteins orchimeric T cell receptor proteins in the same virus/plasmids/T cells asdescribed above or administered to the subject in separateviruses/plasmids/T-cells.

In still another embodiment the methods herein may includeco-administration or a course of therapy also using other small nucleicacid molecules or small chemical molecules or with treatments ortherapeutic agents for the management and treatment of cancer. In oneembodiment, a method of treatment of the invention comprises the use ofone or more drug therapies under conditions suitable for said treatment.

In another embodiment of combination therapy, a passive therapeutic isadministered that can immediately start eliminating the tumor. This isaccompanied by administration of active immunotherapy to induce anactive endogenous response to continue the tumor eradication. In oneembodiment, the methods described herein include administration of theFoxp1-downregulating therapeutic compositions described above with otherknown cancer therapies. For example, surgical debulking, in certainembodiments is a necessary procedure for the removal of large tumormasses, and can be employed before, during or after application of themethods and compositions as described herein. Chemotherapy and radiationtherapy, in other embodiments, bolster the effects of the adoptiveimmunotherapy described herein. Finally, immune-based therapies caneradicate residual disease and activate endogenous antitumor responsesthat persist in the memory compartment to prevent metastatic lesions andto control recurrences. Such combination approaches (surgery pluschemotherapy/radiation plus immunotherapy) are anticipated to besuccessful in the treatment of many cancers along with the methodsdescribed herein.

III. EXAMPLES

The invention is now described with reference to the following examples.These examples are provided for the purpose of illustration only. Thecompositions, experimental protocols and methods disclosed and/orclaimed herein can be made and executed without undue experimentation inlight of the present disclosure. The protocols and methods described inthe examples are not considered to be limitations on the scope of theclaimed invention. Rather this specification should be construed toencompass any and all variations that become evident as a result of theteaching provided herein. One of skill in the art will understand thatchanges or variations can be made in the disclosed embodiments of theexamples, and expected similar results can be obtained. For example, thesubstitutions of reagents that are chemically or physiologically relatedfor the reagents described herein are anticipated to produce the same orsimilar results. All such similar substitutes and modifications areapparent to those skilled in the art and fall within the scope of theinvention.

Example 1: Identification of a New Mechanism of T Cell QuiescenceMediated for Foxp1

We have identified a new mechanism of T cell quiescence mediated byFoxp1, as discussed in Feng, X et al, 2011 Nature Immunol., 12:544-550,incorporated by reference herein. Our data also demonstrate that throughnegative regulation of IL-7 receptor α-chain (IL-7Rα) expression and Tcell receptor (TCR) signaling, Foxp1 is critical in maintaining mature Tcell quiescence. Correspondingly, acute deletion of Foxp1 (all fourisoforms) allows mature naive CD8⁺ T cells to gain effectorphenotypes/functions and to directly proliferate in response to IL-7.

A. Foxp1-Deficient Naive CD8+ T Cells Proliferate in Response to IL-7

To study Foxp1 function in mature T cells, we generatedFoxp1f/fCre-ERT2+RosaYFP mice in which Cre recombinase becomes activatedby treatment with tamoxifen and Cre induces deletion of loxP flankedFoxp1 alleles (Foxp1f/f) and the expression of yellow fluorescentprotein (YFP) as a reporter. We sorted CD44loCD8+ T cells fromFoxp1f/fCre-ERT2+RosaYFP mice and cultured the cells with or withouttamoxifen and IL-7. Without tamoxifen, there was no YFP induction (dataas shown in Feng at FIG. 1a ) or cell proliferation (data not shown) inthe culture by day 6. Without IL-7, most cells died in the culture (datanot shown).

In contrast, in cultures with tamoxifen and IL-7, YFP+ cells started toemerge around day 2 (data not shown). By day 6, in contrast to YFP−cells, most YFP+ cells proliferated, upregulated their expression ofCD44 and CD25, slightly downregulated CD62L and increased in size (dataas shown in Feng at FIG. 1b ). We also observed such phenotypic changes,although to a lesser extent, in nondividing YFP+ cells (data as shown inFeng at FIG. 1b ). We also examined CD44loCD8+ T cells sorted fromFoxp1f/+Cre-ERT2+RosaYFP mice.

Consistent with published observations of only a minor phenotype forCD8+ T cells from Foxp1f/+Cd4-Cre mice (which have one loxPflanked Foxp1allele and expression of Cre driven by Cd4)33,YFP+Foxp1f/+Cre-ERT2+RosaYFP CD8+ T cells still expressed Foxp1 and didnot proliferate or change their phenotype (data not shown). Thisindicated that complete deletion of Foxp1 in CD8+ T cells was requiredfor the phenotypic changes we observed. These changes occurred by day 6only in cultures treated with IL-7, but not in those treated with IL-4(or IL-15; data as shown in Feng at FIG. 1b and data not shown),although there was equally efficient deletion of Foxp1 and induction ofYFP+ cells in all cultures by day 4 (data not shown).

Functional analysis showed that stimulation with phorbol 12-myristate13-acetate (PMA) plus ionomycin induced a greater frequency of IFN-γ-and IL-2-producing YFP+ cells than IFN-γ- and IL-2-producing YFP− Tcells (data as shown in Feng at FIG. 1c ). We also observed suchfunctional changes in nondividing YFP+ cells (data as shown in Feng atFIG. 1c ), which indicated that both the phenotypic changes andfunctional changes in YFP+ cells were induced without proliferation. Theproliferation of YFP+ cells in response to IL-7 was not due tocontamination by CD44hiCD8+ T cells during the sorting of CD44loCD8+ Tcells from the mice, because YFP+ cells from cultures of CD44loCD8+ Tcells proliferated much more than did YFP− cells from cultures of sortedCD44hiCD8+ T cells (data as shown in Feng at FIG. 1b,d ). YFP+ cellsfrom cultures of sorted CD44hiCD8+ T cells also proliferatedsubstantially and upregulated CD25 expression (data as shown in Feng atFIG. 1d ).

To further confirm that Foxp1-deficient naive CD8+ T cells proliferatedin response to IL-7, we sorted CD44loCD8+ T cells fromFoxp1f/fCre-ERT2+RosaYFP mice and control wild-type littermates(Foxp1f/fRosaYFP mice) and cultured them for 2 d, then sorted YFP+ andwild-type control cells and cultured them in equal numbers with IL-7alone. Because we noted deletion of Foxp1 in some YFP− T cells fromFoxp1f/fCre-ERT2+RosaYFP mice after tamoxifen treatment (data notshown), we used cultured wild-type T cells as controls in this set ofexperiments (for the same reason, we used congenic wild-type controls inthe in vivo transfer experiments described below). By day 6, YFP+ cellsproliferated more, which resulted in more total cells and highergranzyme B expression than that of wild-type control cells (data notshown). These results suggest that the deletion of Foxp1 leads maturenaive CD8+ T cells to gain both effector phenotype and function andproliferate in response to IL-7 in the absence of overt TCR stimulation.Mature naive CD4+ T cells in which Foxp1 was acutely deleted gainedeffector phenotype and function as well (albeit to a much lesser extentthan did CD8+ T cells) but did not proliferate in response to IL-7 orIL-4 in vitro (data not shown).

B. Foxp1 Regulates T Cell Quiescence and Homeostasis In Vivo

To examine how Foxp1 regulates naive T cell quiescence and homeostasisin vivo, and to avoid potential secondary effects due to the deletion ofFoxp1 in non-T cells after tamoxifen treatment in vivo, we used anadoptive-transfer model system. We mixed CD44lo CD8+ or CD4+ T cellssorted from Foxp1f/fCre-ERT2+RosaYFP and control CD45.1+ congenicwild-type mice and labeled the cells with the fluorescent dye CellTrace,then transferred them together into intact recipient mice, whichreceived tamoxifen treatment 1 d before the transfer and for the first 3d after transfer. At 8 d after transfer, most of the YFP+ cells in therecipient mice up regulated their expression of IL-7Rα and CD44 but didnot proliferate (data as shown in Feng at FIG. 6a and data not shown). Ahigher percentage of YFP+ cells than wild-type control cells producedIFN-γ and/or IL-2 after stimulation with PMA plus ionomycin (data asshown in Feng at FIG. 6b ).

At 15 d after transfer, the deletion of Foxp1 induced the proliferationof a substantial fraction of CD8+ and CD4+ T cells (data as shown inFeng at FIG. 6c ). In recipient mice that did not receive any tamoxifentreatment, we detected no proliferation of eitherFoxp1f/fCre-ERT2+RosaYFP T cells or wild-type control T cells at 15 dafter transfer (data not shown). These results suggest that Foxp1 iscritical for the maintenance of quiescence in both naive CD8+ and CD4+ Tcells in lympho-replete mice. The proliferation of Foxp1-deficient naiveCD8+ T cells in response to IL-7 in vitro occurred without TCRstimulation (data as shown in Feng at FIG. 1).

To determine whether TCR engagement is important for the regulation ofnaive T cell quiescence and homeostasis by Foxp1 in vivo, we mixedCD44loCD8+ T cells sorted from Foxp1f/fCre-ERT2+RosaYFP and controlCD45.1+ congenic wild-type mice, labeled the cells with CellTracereagent and cultured them for 2 d with tamoxifen in medium. Theseconditions ensured deletion of Foxp1 before the transfer of cells andsubsequent proliferation in the lymphopenic environment of sublethallyirradiated mice deficient in H-2Kb and H-2Db. At 7 d after transfer,whereas most of the wild-type control cells did not proliferate, manymore Foxp1-deficient naive CD8+ T cells proliferated and upregulatedCD44 expression in recipient mice deficient in H-2Kb and H-2Db (data asshown in Feng at FIG. 7a,b ).

We obtained similar results in parallel experiments in which we firsttransferred cells into irradiated mice deficient in H-2Kb and H-2Db,then treated the recipient mice with tamoxifen in vivo (data not shown).

However, in recipient mice deficient in recombination-activating gene 1,Foxp1-deficient CD8+ T cells proliferated only slightly more thanwild-type control cells did (data not shown), which indicated that theenhanced proliferation of Foxp1-deficient CD8+ T cells in lymphopeniawas less notable when complexes of self peptide and majorhistocompatibility complex were available. Nevertheless, inhibition ofIL-7R signaling with antibody to IL-7 and antibody to IL-7R almostcompletely blocked the proliferation of Foxp1-deficient CD8+ T cells inrecipient mice deficient in H-2Kb and H-2Db (data as shown in Feng atFIG. 7c ). These results suggest that in lymphopenic conditions and inthe absence of (or in the presence of considerably less) engagement ofthe TCR with complexes of self peptide and major histocompatibilitycomplex, Foxp1 is essential for naive T cell quiescence and homeostasisand that this regulation is dependent on IL-7-IL-7R.

We have provided direct evidence that Foxp1 has an indispensable role inmaintaining naïve T cell quiescence, in part by repressing IL-7Rαexpression. That view was supported by the following results:Foxp1-deficient naive CD8+ T cells gained effector phenotype andfunction in response to IL-7 but not in response to IL-4, and the amountof IL-7R was critical for the proliferation of Foxp1-deficient CD8+ Tcells in response to IL-7 in vitro. In addition, Foxp1-deficient naiveCD8+ T cells proliferated in vivo in lymphopenic mice deficient in H-2Kband H-2Db in an IL-7-dependent manner.

Here we have shown that Foxp1 is a repressor of IL-7Rα expression. Italso seemed that Foxp1 and Foxo1 had the ability to bind to the samepredicted forkhead-binding site in the Il7r enhancer region, whichsuggests that these two transcription factors may compete for thebinding and antagonize each other to regulate IL-7Rα expression in Tcells. The finding that down regulation of IL-7Rα expression in theabsence of Foxo1 was Foxp1 dependent indicates a potential role of Foxp1in regulating Foxo1 function.

Foxp1 and Foxo1 have been shown to upregulate the expression ofrecombination-activating genes during early B cell development bybinding to the same ‘Erag’ enhancer region. However, neithertranscription factor seems to regulate these genes in T lineage celldevelopment. Foxo1 affects IL-7Rα expression in early B lineage cells,but deletion of Foxp1 does not affect IL-7Rα expression in pro-B cells.These results suggest that Foxp1 and Foxo1 may interact in complex anddistinct ways in different parts of the immune system.

In addition to regulating IL-7Rα expression, Foxp1 seems to controlother molecules critical for regulating T cell quiescence. Our resultshave shown that Foxp1 negatively regulates the MEK-Erk pathway.Transgenic mice expressing the oncogenic protein K-Ras develop T celllymphoma and/or leukemia characterized by aberrantly high CD44expression in thymocytes, which is consistent with the activatedphenotype of Foxp1-deficient thymocytes.

We attempted to introduce a dominant negative K-Ras to inhibit MEK-Erksignaling and determine whether it could restore the activated phenotypeof Foxp1-deficient T cells in Foxp1f/fCd4-Cre mice. However, thedominant negative K-Ras almost completely blocked T cell development(data not shown). Nonetheless, we found that blocking MEK-Erk activationimpaired the proliferation of Foxp1-deficient naive CD8+ T cells inresponse to IL-7 in vitro. It has been shown that the MEK-Erk pathwaycan be regulated by IL-7R and signaling pathways other than TCRsignaling. Because Foxp1-deficient CD8+ T cells in which IL-7Rαexpression was adjusted to nearly wild-type amounts still proliferatedin response to IL-7 (whereas wild-type CD8+ T cells did not), blockingMEK-Erk activation probably inhibits pathways regulated by Foxp1, butindependently of IL-7R-signaling. It has been proposed that constitutivelow-intensity TCR signaling, independently of receptor ligation, has animportant role in T cell development, and low Erk kinase activity ispart of the TCR basal signaling. Therefore, although the proliferationof Foxp1-deficient naive CD8+ T cells in response to IL-7 in vitro andin lymphopenic mice deficient in H-2Kb and H-2Db in vivo would indicatethat there is no obvious TCR engagement, Foxp1 may regulate a basal TCRsignal involving MEK-Erk activity.

It is possible that in the absence of Foxp1, the integration of bothenhanced IL-7R and basal TCR signals act together to drive the naïve Tcells to proliferate. The nature and role of basal TCR signaling inmature T cells remain unknown. Further studies are needed to address howMEK-Erk signaling is involved in the Foxp1 regulation of T cellquiescence in the absence of overt TCR stimulation.

Naive CD4+ T cells with acute deletion of Foxp1 did not proliferate inresponse to IL-7 in vitro. Although the underlying mechanism is notclear, this observation is consistent with published reports showingthat CD8+ T cells are more responsive to IL-7 than CD4+ T cells in invitro cultures9. Nevertheless, we have shown that naive CD4+ T cells inwhich Foxp1 was acutely deleted had proliferation in intact recipientmice similar to that of Foxp1-deficient naive CD8+ T cells, whichindicates that Foxp1 controls T cell quiescence in both CD4+ and CD8+ Tcells in vivo.

In summary, our results have shown that Foxp1 exerts essentialcell-intrinsic transcriptional regulation on the quiescence andhomeostasis of naive T cells by negatively regulating IL-7Rα expressionand MEK-Erk signaling. Our findings have demonstrated coordinatedregulation that actively inhibits T cell activation signals and indicatethat lymphocyte quiescence does not occur by default but is activelymaintained.

Example 2: Wild-Type Tumor-Associated T Cells Up-Regulate Foxp1 mRNA asTumor Progresses

In addition, we have demonstrated that tumor-associated T cellsup-regulate Foxp1 mRNA as tumor progresses in preclinical models ofestablished ovarian carcinoma.

Foxp1 mRNA was quantified by real-time PCR in adoptively transferredCD3⁺CD8⁺ T cells sorted from tumor locations at different temporalpoints after ID8-Vegf/Defb29 tumor challenge. Adoptively transferredeffector T cells represent naïve T cells primed for 7 days against tumorantigen in vitro (Cancer Res. 2009; 69: 6331-8). These T cells wereFACS-sorted from peritoneal wash samples based on the expression of a“congenic” marker (CD45.1), which is not expressed by endogenous(non-transferred, CD45.2) T cells. Relative mRNA expression wasstandardized by GAPDH mRNA.

Immersed in ovarian tumor masses, it is likely that the potentialrepeated tumor antigen stimulation, plus the presence of regulatorydendritic cells (DCs) and alternatively polarized macrophages presentingantigens in the context of suboptimal levels of co-stimulatorymolecules, all promote sustained expression of high levels of Foxp1(particularly Foxp1D), which we have shown to dampen T cell responses.The results are shown in the graph of FIG. 1B.

Example 3: Tumor-Reactive Foxp1-Deficient T Cells Exert SuperiorTherapeutic Effects Compared to Wild-Type T Cells

To further determine the role of Foxp1, we treated a preclinical mousemodel of established ovarian carcinoma, i.e., ID8-Defb29/Vegf-a ovariancarcinoma bearing mice, as described in Conejo-Garcia J R, et al, 2004Nat Med, 10(9):950-8 and Cubillos-Ruiz J R, et al, 2009 J. Clin.Invest., 119(8):2231-44). In this study, mice express a congenic marker(CD45.1), to allow distinguishing adoptively transferred from endogenousT cells in subsequent experiments.

Anti-tumor T cells were negatively immune purified from the spleens ofnaïve (non tumor-bearing) mice, which were either Foxp1-deficient(Foxp1^(f/f)) or wild-type (Foxp1^(f/+)). These mice strains aregenerated as described in Feng, X et al, 2010 Blood, 115(3):510-518,incorporated herein by reference. Both classes of T cells were thenprimed for 7 days against tumor antigens derived from resected ovariantumors of the mouse model, as described in Nesbeth, Y. C. et al. 2010 JImmunol 184, 5654-62, incorporated by reference.

The mice were allowed to grow under normal conditions for 23 days inwhich they developed (advanced) orthotopic ovarian cancer. At day 23,either the Foxp1-deficient T cells or wild type T cells (both of themexpressing CD45.2) were adoptively transferred into separate groups ofmice at 10⁶ anti-tumor T cells/mouse by ip injection.

Peritoneal wash samples were taken from the mice at day 3 and day 7 postadoptive transfer and examined by FACS. FACS analysis (not shown) wasperformed of T cell types in a serum sample subjected to flow cytometryon day 3 and day 7 following the adoptive transfer to theID8-Defb29/Vegf-a mouse model of ovarian cancer of Foxp1-deficient Tcells, showing gating on CD3+ and using a CD45.2 marker, CD8+ marker andCD4+ marker. The results are shown in Table 2 below.

TABLE 2 Foxp1KO T cell WT Control T Day Cell Type Transfer cell Transfer3 # CD45.2⁺ T cells 18876 38980 % Total T cells  6.92% 6.57% # CD8⁺ Tcells/% CD45.2⁺ T 5895/31.2% 7560/19.4% cells # CD4⁺ T cells/% CD45.2⁺ T12095/64.1%  29629/76%   cells 7 # CD45.2⁺ T cells  5656 10450 % Total Tcells 0.996% 1.11% # CD8⁺ T cells/% CD45.2⁺ T 4522/80%   1636/15.7%cells # CD4⁺ T cells/% CD45.2⁺ T  666/11.8% 8592/82.2% cells

The FACS analysis of adoptively transferred lymphocytes at tumor(peritoneal) locations showed that adoptive cell transfer ofFoxp1-deficient T cells in the mice with ovarian cancer produced asignificant increase in survival of CD8+ lymphocytes (cytotoxic effectorT cells) after 3 and 7 days over that seen when the mice wereadministered wild-type T cells. Therefore, Foxp1-deficienttumor-reactive T cells are superior to resist tumor-inducedimmunosuppressive signals and elicit enhanced anti-tumor immunity.

As shown in FIG. 1A, the administration of wild-type tumor-reactive Tcells induced some modest but significant survival increase in theabsence of any host conditioning intervention. In contrast (data notshown), adoptive administration of anti-tumor Foxp1-deficient T cellsstopped ovarian cancer progression, so that all treated mice do not showany signs of disease 6 days after the last mouse treated with controlwild-type T cells died.

Example 4: Generation of siRNA and shRNA

A. Design of an siRNA

The design of suitable siRNA involves the design of the siRNA with 21,23, or 27 nucleotides for modulation of Foxp1, without chemicalmodification. The Foxp1 target gene SEQ ID NO: 1 is screened foraccessible sites and siRNA is synthesized considering the open readingframe (ORF) sequences of Foxp1.

The following general requirements are considered in siRNA design:

-   -   a. No runs of four or more A, T, G, or U in a row.    -   b. The following sequences are avoided, as they can induce an        interferon response. A) 5′-UGUGU-3′ and B) 5′-GUCCUUCAA-3′    -   c. The first 200 bases are omitted from the start codon to avoid        binding to regulatory element of SEQ ID NO: 1.    -   d. Each siRNA duplex is checked in silico to avoid silencing of        off-target effects made on BLAST search considering the        following parameters:        -   I. Low complexity filtering is removed to avoid            insignificance by BLAST resulting in limited or no query            sequencer.        -   II. The word size was set to 7 letters, the minimal value            algorithm        -   III. Expected value threshold is set at 1000 to avoid the            probability of short sequence occurrence.            -   siRNA synthesis is performed by commercially available                methods (e.g., Qiagen) using chemically-protected                phosphoramidite monomers. Resultant oligomers are                purified by PAGE, desalting, or IE-HPLC. The quality of                each siRNA is analyzed by MALDI-TOF and yields are                determined by an integrated spectrophotometer.

One embodiment of an siRNA for Foxp1 has the sequence:

SEQ ID NO: 3 AAUCUGGGACUGAGACAAA.

Another embodiment of an siRNA for Foxp1 has the sequence:

SEQ ID NO: 4 GAUGCAAGAAUCUGGGACU.

B. Design of an siRNA Nanoparticle

The siRNA in one embodiment is conjugated with PEI by conventionalmethods, for example as described in Cubillos-Ruiz et al, J. Clin.Invest., 119(8):2231-44 (August 2009) which is incorporated by referenceherein.

C. Design of an shRNA

The shRNA is based upon the siRNA sequence above or the ORF of Gene ID:27086 (see SEQ ID NOs: 1 and 2). Using commercial available methods, theshRNA is designed by synthesizing the siRNA sequence above, followed bya 9-15 nt loop sequence, and further followed by a reverse complement ofthe siRNA sequence. For examples, see, e.g., the DNA sequences that areused to express in vectors the shRNAs recited in Table 1.

Example 5: Designing a Virus Containing a Foxp1 shRNA and Preparation ofAnti-Tumor T Cells

To achieve Foxp1 knock-down in a therapeutic setting in humans, we useFoxp1 shRNA-containing Lentivirus that are currently being used inclinical trials for gene therapy. For example, we use aself-inactivating lentiviral vector (GeMCRIS 0607-793) which wassuccessfully used to transduce T cells directed against tumor cells inleukemia patients (Porter et al., N Engl J Med. 2011 Aug. 25;365(8):725-33). By using this lentiviral vector and transductions at amultiplicity of infection of 5, a high level of expression of chimericreceptors directed against tumor cell antigens can be obtained in >85%primary human T cells (Milone et al., Molecular Therapy (2009) 17(8),1453-64).

A cassette containing a Foxp1 shRNA sequence downstream of a RNApolymerase III promoter (e.g., the H1 or the U6 promoter) is subclonedinto pRRL-SIN-CMV-eGFP-WPRE, a third generation, self-inactivatinglentiviral vector plasmid, replacing the CMV promoter. The presence ofthis single construct confers expression of the chimeric receptor andsilencing of Foxp1 in the same T cell. Alternatively, different viralvectors are used to express chimeric receptors and shRNA. In anotherexperiment, polyclonal T cells primed against multiple tumor antigensare transduced only with this lentiviral vector encoding a Foxp1 shRNAsequence. The Foxp1 shRNA sequences that are used are listed in Table 1.

Thereafter we infect purified human T lymphocytes and examine theexpression levels of Foxp1 by western blotting. The cells that achieveefficient Foxp1 knock-down are used later in a therapeutic setting.

Example 6: T Cell Mediated Immunotherapy in an Ovarian Cancer Model

Mice: Female B6.Ly5.2 (congenic, CD45.1+) mice were received fromNational Cancer Institute. Foxp1^(f/f)CD4-Cre (Foxp1 KO) and controlFoxp1^(+/+) (Control Foxp1^(+/+)) mice in CD45.2+ background were bredin the animal facility of the Wistar Institute, Philadelphia. Allexperiments were performed in accordance with the protocols approved byInstitutional Animal Care and User Committee of the Wistar Institute.

Ovarian Tumor Model: We initially used transplantable ID8 mice model forovarian cancer. Preliminary experiments utilize modification of ID8,ID8-Vegf/Defb29 which is more aggressive and recapitulates themicroenvironment of human solid ovarian tumors. Moreover, compared tothe parental ID8 model, immune rejection of established ID8-Vegf/Defb29tumors are not yet reported.

Generation of ovarian tumors in mice: On day 0 of the experiments, 2×10⁶ID8-Vegf/Defb29 cells were intraperitoneally inoculated into 6-7 weekold female mice.

Example 7: In Vitro Priming of Foxp1 KO and Control Foxp1^(+/+) T Cellsfor Immunotherapy

A. Generation of Murine Bone Marrow Derived Dendritic Cells (BMDCs) forIn Vitro T Cell Priming.

BMDCs were generated from bone marrow (BM) collected from tibia andfemur of 6-8 week old female C57Bl/6 mice. 2×10⁶ BM cells were culturedin 10 ml RPMI1640 medium supplemented with 10% heat inactivated fetalbovine serum, penicillin/streptamicine, 50 mM 2-mercaptoethanol,L-Glutamine and sodium pyruvate (R10 medium) and 10 ng/ml recombinantmouse granulocyte/macrophage-colony stimulating factor (GM-CSF)(Peprotech). Half-medium changes were carried out every two days. On day7, approximately 80% of the cells in culture were CD11c+ dendriticcells.

B. Irradiation of ID8-Vegf/Defb29 Cells:

ID8-Vegf/Defb29 cells were g-irradiated for 10000 rads using a Mark 1irradiator (J. L. Shepherd & Associates, Glendale, Calif.). Cells werethen UV irradiated for 30 minutes.

C. Tumor Antigen Pulsing of BMDCs:

On day 7 of the culture, BMDCs were collected by vigorous pipetting.BMDCs were then cultured over night with irradiated ID8-Vegf/Defb29cells at 10:1 ratio in complete R10 medium. Tumor antigen pulsed BMDCswere recovered on next day for T cell priming.

D. Isolation and In Vitro Priming of Foxp1 KO and Control Foxp1^(+/+) TCells:

T cells were isolated from the spleen and lymph nodes of Foxp1 KO andcontrol Foxp1^(+/+) mice using Militenyi mouse Pan T cell isolation kit.Isolated T cells were cultured for 7 days with tumor antigen pulsedBMDCs at 10:1 ratio in R10 medium supplemented with 50 U/ml IL-2 in a 12well tissue culture plate at a density of 2×10⁶ cells/well.

Example 8: T Cell Immunotherapy with Antigen Primed Foxp1 KO T Cells

On day 7, primed T cells were recovered, and washed with PBS. 1×10⁶primed T cells were intraperitoneally transferred into day 24ID8-Vegf/Defb29 tumor bearing mice (see Example 6). Control micereceived either primed control Foxp1^(+/+) T cells or PBS.

Follow cytometry analysis for cell number and viability: On day 3 and 6of T cell adoptive transfer, two mice each from the Foxp1 KO, controlFoxp1^(+/+) T cell transferred or PBS treated groups were sacrificed. Aperitoneal wash is preformed to collect cells. Spleens and BM were alsocollected. RBC was lysed followed by incubation with anti-CD16/CD32antibody to block nonspecific FcR binding. Cells were stained withCD45.2 (congenic marker), CD4, CD8, CD3e, CD45, 7AAD (viability marker),AnnexinV (apoptosis marker) and analyzed using Becton Dickinson (BD)LSR. Data were processed using FlowJo software.

Survival: ID8-Vegf/Defb29 tumor bearing mice received T cellimmunotherapy or the control PBS on day 24 were closely monitored forsurvival.

Example 9: Use of siRNA and shRNA in Mouse Model of Ovarian Cancer

A. Use of shRNA

About 10⁶ cells/mouse of the T cells of Example 5 are administered tomice of the ovarian cancer model of Example 6 by intraperitonealinjection. Controls are wildtype T cell-injected mice. In differentgroups of treated mice, samples of the blood and peritoneal wash of eachmouse are assayed by FACS after 3, 7 and 21 days. The adoptive T celltherapy using T cells transduced with a lentivirus expressing this shRNAis able to inhibit tumor growth and cause tumor regression in thisanimal model. Therefore the present compositions and methods aresuitable and effective for therapy of cancer and proliferative disease.

B. Use of siRNA

About 50 μg/injection of the siRNA/siRNA nanoparticle described abovewas administered to mice of the ovarian cancer model of Example 2 byintraperitoneal injection. Controls are PBS-injected mice. In differentgroups of treated mice, samples of the blood and peritoneal wash of eachmouse is assayed by FACS after 3, 7 and 21 days. A significant increasein survival was observed.

The siRNA composition is able to inhibit tumor growth and cause tumorregression in these animal models. Therefore the present compositionsand methods are suitable and effective for therapy of cancer andproliferative diseases.

Example 10: Foxp1 is Up-Regulated in Tumor-Reactive T Cells in the TumorMicroenvironment

Naïve, negatively immunopurified T cell splenocytes were primed againsttumor antigen for 7 days by incubation with bone marrow-deriveddendritic cells pulsed with double (UV+gamma) irradiated tumor cells(FIG. 2, lanes 1 and 3; Day 6 effectors). Tumor antigen-primed T cellswere then administered to CD45.1 (congenic) advanced ID8-Defb29/VEGFαtumor-bearing mice. Mice were sacrificed 2 days later and adoptivelytransferred (CD45.2+) CD8 and CD4 T cells were FACS-sorted fromperitoneal wash samples (FIG. 2, lanes 2 and 5; Day 2 after transfer),along with endogenous (host-derived, CD45.1) tumor-associated T cells(FIG. 2, lanes 3 and 6). Foxp1 was quantified in all samples byWestern-blot.

Example 11: Foxp1 T Cells are Resistant to the Abrogation of T CellExpansion Induced by TGFβ

Naïve, negatively immunopurified CD8 and CD4 T cell splenocytes (fromboth wild-type and Foxp1 KO mice) were CFSE-labeled and expanded in thepresence of CD3/CD28 Ab-coated beads (Invitrogen). When indicated, 5ng/mL of TGFb was added, and CFSE dilution (indicative of T cellproliferation) was monitored by flow cytometry. The results ofabrogation of T cell expansion induced by TFGβ are shown by contrastingFIG. 3A with 3C and 3B with 3D.

Example 12: TGFβ Repress cJun Activation in Wt but not in Foxp1 KO Cd8+T Cells

Naïve, negatively immunopurified CD8 T cell splenocytes (from bothwild-type and Foxp1 KO mice) were stimulated in the presence of CD3/CD28Ab-coated beads (Invitrogen). When indicated, 5 ng/mL of TGFb was addedto the wells, and both total and phosphorylated c-Jun were analyzed byWestern-blot. Note the induction of c-Jun phosphorylation upon CD3/CD28activation and how it is selectively abrogated in wild-type T cells inthe presence of TGFβ.

Examples 10-12 show that the Foxp1 and TGFβ pathways converge, so thatFoxp1 is required for the tolerogenic effect induced by TGFβ in themicroenvironment of virtually all solid tumors. Correspondingly,Foxp1-deficient tumor reactive T cells are insensitive to thisimmunosuppressive mechanism, and therefore exert superior antitumorprotection. In addition, Foxp1 is upregulated in T cells in the tumormicroenvironment at the protein level, pointing to a mechanism oftumor-induced immunosuppression.

Each and every patent, patent application including U.S. patentapplication Ser. No. 14/350,588, International Patent Application No.PCT/US2012/061556, and U.S. Provisional patent application No.61/552,630, and publication, including publications listed herein, andpublically available peptide sequences cited throughout the disclosure,as well as the Sequence Listing, is expressly incorporated herein byreference in its entirety. Embodiments and variations of this inventionother than those specifically disclosed above may be devised by othersskilled in the art without departing from the true spirit and scope ofthe invention. The appended claims include such embodiments andequivalent variations.

The invention claimed is:
 1. A composition for treating a cancercomprising a T cell pulsed ex vivo with a selected cancer-specificantigen or tumor-specific antigen, the T cell having therein a vectorexpressing a short hairpin RNA (shRNA) construct that down regulates orextinguishes Foxp1, the shRNA under the control of a suitable promoter,which composition is in a pharmaceutically acceptable carrier or diluentthat is suitable for administration to a human subject, wherein theshRNA is expressed in the vector by a DNA sequence of SEQ ID NO:
 5. 2.The composition according to claim 1, wherein the vector is a viralvector selected from the group of lentiviral, adenoviral or retroviralvectors.
 3. The composition according to claim 1, wherein said antigenis from a cancer or tumor which is selected from the group consisting ofbreast cancer, lung cancer, prostate cancer, colorectal cancer, braincancer, esophageal cancer, stomach cancer, bladder cancer, pancreaticcancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma,leukemia, myeloma, lymphoma, glioma, and multidrug resistant cancer. 4.The composition according to claim 2, wherein the shRNA containingvector is a lentiviral vector and the promoter is an RNA polymerase IIIpromoter.
 5. A composition for treating a cancer comprising a T cellpulsed ex vivo with a selected cancer-specific antigen or tumor-specificantigen, the T cell and having therein a vector expressing a shorthairpin RNA (shRNA) construct that down regulates or extinguishes Foxp1,the shRNA construct under the control of a suitable promoter, whichcomposition is in a pharmaceutically acceptable carrier or diluent thatis suitable for administration to a human subject, wherein the shRNA isexpressed in the vector by a DNA sequence of SEQ ID NO: 8, 11 or
 14. 6.The composition according to claim 5, wherein the vector is a viralvector selected from the group of lentiviral, adenoviral or retroviralvectors.
 7. The composition according to claim 5, wherein said antigenis from a cancer or tumor which is selected from the group consisting ofbreast cancer, lung cancer, prostate cancer, colorectal cancer, braincancer, esophageal cancer, stomach cancer, bladder cancer, pancreaticcancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma,leukemia, myeloma, lymphoma, glioma, and multidrug resistant cancer. 8.The composition according to claim 5, wherein the shRNA containingvector is a lentiviral vector and the promoter is an RNA polymerase IIIpromoter.